Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells
In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically mo...
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MDPI AG
2020-06-01
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| Series: | Animals |
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| Online Access: | https://www.mdpi.com/2076-2615/10/6/1078 |
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| author | Ana Carolina Furlanetto Mançanares Joel Cabezas José Manríquez Vanessa Cristina de Oliveira Yat Sen Wong Alvaro Daniela Rojas Felipe Navarrete Aguirre Lleretny Rodriguez-Alvarez Fidel Ovidio Castro |
| author_facet | Ana Carolina Furlanetto Mançanares Joel Cabezas José Manríquez Vanessa Cristina de Oliveira Yat Sen Wong Alvaro Daniela Rojas Felipe Navarrete Aguirre Lleretny Rodriguez-Alvarez Fidel Ovidio Castro |
| author_sort | Ana Carolina Furlanetto Mançanares |
| collection | DOAJ |
| description | In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells. |
| first_indexed | 2024-03-10T18:57:20Z |
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| language | English |
| last_indexed | 2024-03-10T18:57:20Z |
| publishDate | 2020-06-01 |
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| series | Animals |
| spelling | doaj.art-7354a6f8427b4693b5a76b6fe1be6d862023-11-20T04:39:08ZengMDPI AGAnimals2076-26152020-06-01106107810.3390/ani10061078Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem CellsAna Carolina Furlanetto Mançanares0Joel Cabezas1José Manríquez2Vanessa Cristina de Oliveira3Yat Sen Wong Alvaro4Daniela Rojas5Felipe Navarrete Aguirre6Lleretny Rodriguez-Alvarez7Fidel Ovidio Castro8Department of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Campus Chillan, Chillán 3780000, ChileDepartment of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Campus Chillan, Chillán 3780000, ChileDepartment of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Campus Chillan, Chillán 3780000, ChileDepartment of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo, Pirassununga, São Paulo 13630-000, BrazilDepartment of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Campus Chillan, Chillán 3780000, ChileDepartment of Animal Pathology, Faculty of Veterinary Sciences, Universidad de Concepción, Campus Chillan, Chillán 3780000, ChileDepartment of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Campus Chillan, Chillán 3780000, ChileDepartment of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Campus Chillan, Chillán 3780000, ChileDepartment of Animal Science, Faculty of Veterinary Science, Universidad de Concepción, Campus Chillan, Chillán 3780000, ChileIn mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.https://www.mdpi.com/2076-2615/10/6/1078CRISPR/cas9prostaglandin E2adipose mesenchymal stem cellequine |
| spellingShingle | Ana Carolina Furlanetto Mançanares Joel Cabezas José Manríquez Vanessa Cristina de Oliveira Yat Sen Wong Alvaro Daniela Rojas Felipe Navarrete Aguirre Lleretny Rodriguez-Alvarez Fidel Ovidio Castro Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells Animals CRISPR/cas9 prostaglandin E2 adipose mesenchymal stem cell equine |
| title | Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells |
| title_full | Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells |
| title_fullStr | Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells |
| title_full_unstemmed | Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells |
| title_short | Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells |
| title_sort | edition of prostaglandin e2 receptors ep2 and ep4 by crispr cas9 technology in equine adipose mesenchymal stem cells |
| topic | CRISPR/cas9 prostaglandin E2 adipose mesenchymal stem cell equine |
| url | https://www.mdpi.com/2076-2615/10/6/1078 |
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